Page 200 - Haematologica Vol. 109 - July 2024

ARTICLE - FLT3L promotes osteolysis in multiple myeloma
D. Shin et al.
with FLT3L-DKK1 pathway-mediated bone osteolysis.
To understand the characteristics of the HY subtype, we next identified 662 genes predominantly up-regulated in the HY subtype (Online Supplementary Figure S5A) and then examined cellular pathways represented by these genes using ConsensusPathDB31 (Figure 4C). These genes defining the HY subtype were significantly (P<0.05) associated with FLT3 and JAK-STAT signaling pathways, consistent with our
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findings (see Figure 2). We further divided the HY subtype patients into those with high levels of both FLT3L and DKK1 (FLT3LHDKK1H in Figure 4B) and the others (FLT3LHDKK1L, FLT3LLDKK1H and FLT3LLDKK1L), and identified 984 differ- entially expressed genes (DEG; 471 up-regulated and 513 down-regulated in FLT3LHDKK1H) between the 2 subgroups. The up-regulated genes most strongly represented cytokine response, and also, significantly, other pathways previously
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Figure 3. Fms-like tyrosine kinase 3 ligand-induced Dick Kopf-related protein 1 attenuates WNT/b-catenin signaling. (A) Condi- tioned media (CM) obtained from HEK293T transiently transfected with DNA construct, such as Dick Kopf-related protein 1 (DKK1), Fms-like tyrosine kinase 3 ligand (FLT3L), or both were treated into newly HEK293T cells for three days. Expression of b-catenin protein from cell lysates was analyzed by western blot assay. (B) EV (empty vector) and DKK1 transiently expressing HEK293T cells were treated with rhFLT3L (40 ng/mL) for three days. Nuclear and cytoplasmic fraction from the cell pellets was separated by Extraction kit. Protein levels of b-catenin and GAPDH (cytosol) and Lamin A/C (Nucleus) as loading controls were determined by western blotting. (C) Reporter gene assay for b-catenin-mediated transcriptional activation was conducted in HEK293T transient- ly transfected with plasmids carrying EV or DKK1 under treatment of rhFLT3L (40 ng/mL) for three days (N=4). (D) Alkaline phos- phatase staining and (E) activity were evaluated with MC3T3-E1 cells treated with mock, rhFLT3L, rhBMP2 or rhDKK1 for seven days (N=6). (F) Relative mRNA expression level of DKK1 was determined by qRT PCR. mRNA was prepared from the MC3T3-E1 cells treated with mock, rhFLT3L, rhBMB2 or rhDKK1 for seven days (n=3). Error bars indicate the standard deviation of at least 3 in- dependent replicates. ALP: alkaline phosphatase **P<0.01, ***P<0.001, ****P<0.0001, ns: not significant (P>0.05).
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